U.S. patent application number 11/595291 was filed with the patent office on 2007-08-02 for method and means for treating solid tumors.
Invention is credited to Essam T. Awdalla.
Application Number | 20070178107 11/595291 |
Document ID | / |
Family ID | 46326553 |
Filed Date | 2007-08-02 |
United States Patent
Application |
20070178107 |
Kind Code |
A1 |
Awdalla; Essam T. |
August 2, 2007 |
Method and means for treating solid tumors
Abstract
The present invention relates to method and means for treating a
solid tumor using a number of, in vitro prepared, anticellular
agent(s)-carrying blood platelets to induce a thrombus formation
within the tumor vasculature, and at the same time to deliver a
high concentration of an anticellular agent within the tumor. The
blood platelets are targeted and attached to the tumor vasculature
using in vivo assembled binding complexes, each having at least one
binding site specifically binding to tumor cells or to
tumor-associated vasculature, and at least one binding site
specifically binding to a blood platelet surface. The
platelet-mediated thrombus formed within the tumor vasculature
leads to occlusion of the tumor vasculature, with ultimate
destruction of the centrally located tumor cells. This is followed
by destruction or impairing the growth or cell division of the
peripherally located tumor cells, by the anticellular agent(s)
carried by the blood platelets.
Inventors: |
Awdalla; Essam T.;
(Morrisville, NC) |
Correspondence
Address: |
Essam T. Awdalla
124 Black Ridge Street
Morrisville
NC
27560
US
|
Family ID: |
46326553 |
Appl. No.: |
11/595291 |
Filed: |
November 10, 2006 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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11399281 |
Apr 6, 2006 |
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11595291 |
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11343694 |
Jan 31, 2006 |
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11399281 |
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Current U.S.
Class: |
424/155.1 ;
424/178.1; 514/13.3; 514/13.7; 514/13.8; 514/14.9; 514/171;
514/19.1; 514/19.3; 514/27; 514/283; 514/3.2; 514/34; 514/393;
514/44R; 514/453; 514/54; 514/56; 514/731 |
Current CPC
Class: |
A61K 31/727 20130101;
A61K 31/57 20130101; A61K 31/4745 20130101; A61K 31/704 20130101;
A61K 48/00 20130101; A61K 31/7048 20130101; A61K 31/366
20130101 |
Class at
Publication: |
424/155.1 ;
424/178.1; 514/731; 514/54; 514/56; 514/44; 514/12; 514/393;
514/171; 514/34; 514/283; 514/27; 514/453 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61K 48/00 20060101 A61K048/00; A61K 31/7048 20060101
A61K031/7048; A61K 31/704 20060101 A61K031/704; A61K 31/57 20060101
A61K031/57; A61K 31/4745 20060101 A61K031/4745; A61K 31/366
20060101 A61K031/366 |
Claims
1. In a mammal, a method for treating a vascularized solid tumor,
including the steps of: a) parenteral administration of a
sub-therapeutic dose of at least one small molecular Vascular
Disrupting Agent (VDA), to disrupt the endothelial lining of the
tumor vasculature, while maintaining adequate rate of blood flow
within the tumor vasculature, and thus directly exposing the
underlying tumor cells to the circulating blood; b) Targeting a
number of, in vitro prepared, anticellular agent-carrying blood
platelets to the tumor cells exposed in step (a), leading to
immobilization of the said anticellular agent-carrying blood
platelets within the tumor vasculature; c) allowing for the
induction of a thrombus formation within the tumor vasculature,
leading to occlusion of the tumor vasculature and destruction of
the centrally located tumor cells, followed by rupture of the said
anticellular agent-carrying blood platelets, with release of their
anticellular agent content; and d) arranging for the delivery of
the anticellular agent released from the ruptured blood platelets
in step (c), to the peripherally located tumor cells.
2. In a mammal, a method for treating a vascularized solid tumor,
including the steps of: a) parenteral administration of a
sub-therapeutic dose of at least one small molecular Vascular
Disrupting Agent (VDA), to disrupt the endothelial lining of the
tumor vasculature, while maintaining adequate rate of blood flow
within the tumor vasculature, and thus directly exposing the
underlying tumor cells to the circulating blood; b) parenteral
administration of a number of at least one type of anti-tumor
binding component--first ligand complexes; c) parenteral
administration of a number of anti-ligands; d) parenteral
administration of a number of, in vitro prepared, blood platelets,
each blood platelet carrying at least one anticellular agent and
having at least one anti-platelet binding component--second ligand
complex attached to its outer surface; e) allowing the blood
platelets to link to the tumor vasculature through in vivo
formation of anti-tumor binding component--first
ligand--anti-ligand--second ligand--anti-platelet binding component
complexes; f) parenteral administration of a number of
anti-ligands; g) allowing more anticellular agent-carrying blood
platelets to link to the blood platelets already linked to the
tumor vasculature through in vivo formation of anti-platelet
binding component--second ligand--anti-ligand--second
ligand--anti-platelet binding component complexes, thereby allowing
for the formation of a blood thrombus within the tumor vasculature,
leading to occlusion of the tumor vasculature and destruction of
the centrally located tumor cells, followed by rupture of the
anticellular agent-carrying blood platelets included within the
formed blood thrombus, with release of their anticellular agent
content; and h) encouraging the mammal to exercise few times a day,
for several days, thereby evenly dispersing the anticellular agent
released from the ruptured blood platelets within the debris of the
centrally located tumor cells, which enables delivering the
anticellular agent to the peripherally located tumor cells.
3. The method of claim 2, wherein the formation of a blood thrombus
within the tumor vasculature in step (g) is followed by removal of
the freely circulating residual portion of the administered
anticellular agent-carrying blood platelets, which were not
included within the thrombus formed within the tumor vasculature,
from the mammal's blood stream.
4. The method of claim 2, wherein at least one of the said steps of
the method is preceded and/or accompanied by the administration of
at least one anti-coagulant agent to the mammal.
5. The method of claim 2, which is preceded and/or accompanied by
the administration of at least one immuno-suppressive agent to the
mammal.
6. The method of claim 2, which is preceded and/or accompanied
and/or followed by enteral or parenteral administration of a
therapeutic dose of at least one anticellular agent.
7. The method of claim 2, wherein the small molecular Vascular
Disrupting Agent (VDA) is selected from the group consisting of the
microtubulin destabilizing drugs, combretastatin A-4 disodium
phosphate, ZD6126, AVE8062, and Oxi 4503; and the flavonoid,
DMXAA.
8. The method of claim 2, wherein the anti-tumor binding component
has a binding region specifically binding to an antigen or a
receptor present on the outer surface of a tumor cell, or present
on the outer surface of a component of a tumor associated
vasculature or stroma, with said anti-tumor binding component being
selected from the group consisting of an antibody, a monoclonal
antibody, a polyclonal antibody, a humanized monoclonal antibody, a
chimeric antibody, a single chain antibody, a dimeric single chain
antibody construct, a multimeric single chain antibody construct, a
peptide, a nucleic acid sequence, a protein, a ligand or
anti-ligand, an oligonucleotide, native or naked antibodies;
chimeric monoclonal antibodies; genetically engineered monoclonal
antibodies; fragments of antibodies, tumor-binding peptides;
polypeptide; glycoprotein; lipoprotein, growth factors; lymphokines
and cytokines; enzymes, immune modulators; fusion protein,
enzymatic substrate, receptor, hormone, lectin, cadherin,
immunological conjugates, chemical conjugates, any of the above
joined to a molecule that mediates an effector function; conjugates
that include any one of the above; and fragments or parts of any of
the above.
9. The method of claim 2, wherein at least two types of anti-tumor
binding components are used, with at least one of them specifically
binding to an antigen or a receptor present on the outer surface of
a tumor cell, and at least another one of them specifically binding
to an antigen or a receptor present on the outer surface of a
component of a tumor associated vasculature or stroma.
10. The method of claim 2, wherein the first and second ligands and
the anti-ligand consist of a complementary set of molecules that
specifically bind to each other and are selected from the group
consisting of biotin/avidin or streptavidin or a chemically
modified form of streptavidin or avidin; zinc finger protein/dsDNA
fragment; enzyme/inhibitor; hapten/antibody; ligand/receptor;
homophylic peptides; and leucine zipper sets.
11. The method of claim 2, wherein the anti-platelet binding
component has a binding region specifically binding to an antigen
or a receptor present on the outer surface of the blood platelet
and is selected from the group consisting of an antibody, a
monoclonal antibody, von Willebrand factor, osteopontin,
fibrinogen, fibrin, fibronectin, vitronectin, collagen,
thrombospondin, laminin, heparin, heparan sulfate, chondroitin
sulfate, phospholipase A2, matrix metalloproteinases, thrombin,
glass, sialyl-lewis X, fibulin-1, PECAM, ICAM-1, ICAM-2, p-selectin
ligand, MAC-1, LFA-1, portions of any of the above, and functional
equivalents of any of the above.
12. The method of claim 2, wherein the blood platelets are freshly
isolated platelets.
13. The method of claim 2, wherein the blood platelets are
rehydrated fixed-dried platelets.
14. The method of claim 2, wherein the anticellular agent is
selected from the group consisting of radioactive isotopes,
cytotoxins, chemotherapeutic agents, steroids, antimetabolites,
anthracyclines, vinca alkaloids, antibiotics, alkylating agents,
epipodophyllotoxins, and any plant-, fungus- or bacteria-derived
agent.
15. The method of claim 2, wherein the anticellular agent is
contained within the blood platelet.
16. The method of claim 2, wherein the anticellular agent is
attached to the outer surface of the blood platelet.
17. The method of claim 2, wherein more than one anticellular agent
are used, with at least one anticellular agent being contained
within the blood platelet, and at least another anticellular agent
being attached to the outer surface of the blood platelet.
18. The method of claim 2, wherein the mammal is a human cancer
patient.
19. In a mammal affected by multi-focal, variable-sized, secondary
metastases deposits of a solid tumor, with at least one of the
secondary metastases deposits being vascularized, and at least
another one of the secondary metastases deposits being not-yet
vascularized, a schedule for treating said solid tumor secondary
metastases deposits including the steps of: a) treating the mammal
using the method of claim 2, to destroy the vascularized secondary
metastases deposit(s) of the tumor; b) administration of at least
one agent to prevent/minimize the implantation of new tumor
secondary metastases deposits within the mammal's body, for a
period of time sufficient for the not-yet vascularized secondary
metastases deposit(s) of the tumor to grow and develop its own
vasculature; and c) re-treating the mammal using the method of
claim 2, to destroy the now vascularized secondary metastases
deposit(s) of the tumor, and thus, completely clearing the
secondary metastases deposits of the tumor from the mammal's
body.
20. The method of claim 19, wherein the administered agent(s) to
prevent/minimize the implantation of new tumor secondary metastases
deposits within the mammal's body is selected from the group
consisting of anti-coagulants; platelet inhibitors;
thrombocytopenic agents; and vascular repairing agents.
21. The method of claim 19, wherein the mammal is a human cancer
patient.
Description
CROSS-REFERENCE TO RELATED APPLICATION(S)
[0001] This non-provisional utility patent application claims the
benefit of one prior filed co-pending non-provisional patent
application; the present application is a continuation-in-part of
U.S. patent application Ser. No. 11/399,281, filed Apr. 6, 2006,
which is a continuation-in-part of U.S. patent application Ser. No.
11/343,694, filed Jan. 31, 2006, which are incorporated herein by
reference in their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates generally to a method for
treating solid tumors in a mammal, and more particularly to a
method for treating a vascularized solid tumor by targeting a
plurality of anticellular agent-carrying blood platelets to the
vasculature of the tumor, to induce a thrombus formation within the
tumor vasculature, and at the same time to deliver a high
concentration of the anticellular agent to the tumor cells, and
thus, clearing solid tumors entirely from the mammalian body.
BACKGROUND OF THE INVENTION
[0003] Cancer represents a group of diseases characterized by
uncontrolled growth and proliferation of abnormal cells, which, if
not controlled, results in death of the host. Cancer continues to
be one of the most serious diseases threatening human and animal
health and life. The American Cancer Society estimated that about
1,372,910 new cancer cases were diagnosed in the USA in 2005, with
solid tumor cases accounting for more than 90% of all cancer cases.
In the same year, 570,280 cancer patients were expected to die in
the USA. These statistics translate to more than 1600 deaths per
day. Cancer is the second leading cause of death in the USA next to
the coronary heart diseases.
[0004] The traditional treatment of cancer patients involves a
combination of surgery, radiotherapy and/or chemotherapy,
unfortunately, combined treatment with all three modalities have
not shown to be effective against all cancer and tumor cells, due
to the wide heterogeneity of cancer cells regarding their
metabolism, enzyme composition, growth rate and gene errors, with
some of the cancer cells being usually resistant to each of the
used treatment modalities. The resistant cells survive, seed, and
continue to grow in the living host, with subsequent treatments
being less effective at killing the cancer cells.
[0005] As a solid tumor grows, in order to sustain itself, it must
develop its own blood supply. This blood supply, however, is much
different from the blood supply to normal tissues. The blood
vessels formed in tumors are typically highly irregular and
tortuous. They may have arterio-venous shunts and blind ends, and
lack smooth muscle or nerves and have incomplete endothelial
linings and basement membranes. This leads to low overall levels of
oxygen in most tumors. Many tumors have areas of extreme hypoxia.
(Brown, J. M. "Exploiting the hypoxic cancer cell: mechanisms and
therapeutic strategies" Molecular Medicine Today, April 2000 (Vol.
6)). Such hypoxic areas are known to be refractory towards many of
the currently available treatments for solid tumor cancers,
including radiation therapy and chemotherapy.
[0006] Several unconventional approaches for treating solid tumors
are being proposed continuously, with targeting the tumor
vasculature with Vascular disrupting agents (VDAs) being the most
promising of these approaches. Vascular disrupting agents (VDAs)
are designed to cause a rapid and selective shutdown of the blood
vessels of tumors. Unlike antiangiogenic drugs that inhibit the
formation of new vessels, VDAs occlude the pre-existing blood
vessels of tumors to cause tumor cell death from ischemia and
extensive hemorrhagic necrosis. There are broadly two types of
VDAs, small molecules and ligand-based, which are grouped together,
because they both cause acute vascular shutdown in tumors leading
to massive tumor necrosis. The small molecule VDAs include the
microtubulin destabilizing drugs, combretastatin A-4 disodium
phosphate, ZD6126, AVE8062, and Oxi 4503, and the flavonoid, DMXAA.
Ligand-based VDAs use antibodies, peptides, or growth factors that
bind selectively to tumor blood vessels to target tumors with
agents that occlude blood vessels. The ligand-based VDAs include
fusion proteins (e.g., vascular endothelial growth factor linked to
the plant toxin gelonin), immunotoxins (e.g., monoclonal antibodies
to endoglin conjugated to ricin A), antibodies linked to cytokines,
liposomally encapsulated drugs, and gene therapy approaches.
[0007] However, as VDAs are designed to occlude the tumor
vasculature, so they cut off the blood supply to only the centrally
located tumor cells, which rely on the tumor vasculature for their
nutrition, leading to their destruction, while sparing the
peripherally located outer rim of tumor cells, which rely on the
surrounding blood vessels and interstitial fluids for their
nutrition. The surviving outer rim of tumor cells will eventually
grow and replace the destroyed central part of the tumor. Thus
there is a need for another VDAs-like approach for treating solid
tumors, with which both the centrally located and the peripherally
located tumor cells may be simultaneously destroyed.
[0008] Prior art documents include components related to the field
of the present invention but lack an integrated, combined solution
that is provided by the present invention, including the
following:
Targeting antibodies carrying diagnostic or therapeutic agents to
the vasculature of solid tumor masses, through recognition of tumor
vasculature-associated antigens described by Thorpe et al in U.S.
Pat. Nos. 5,776,427, 5,863,538; delivering a compound of interest
to a thrombogenic surface, using fixed-dried blood platelets
carrying said compound, as described by Nichols, Timothy C.; et al.
in U.S. patent application Ser. No. 11/149,515; monoclonal
antibodies and their fragments, which may be derived from any
species (including humans) or may be formed as chimeric proteins
which employ sequences from more than one species, using
conventional techniques, such as hybridoma synthesis, recombinant
DNA techniques and protein synthesis. See, generally, Kohler and
Milstein, Nature, 256: 495-97, 1975; and Eur. J. Immunol., 6:
511-19, 1976; both of which are incorporated herein by reference;
human, or humanized, monoclonal antibodies recognizing surface
antigens of cancer cells. Non limiting examples are described by
Hosokawa, et al. in U.S. Pat. No. 6,787,153, by Taniguchi, et al.
in U.S. Pat. No. 4,800,155, by Abe, et al. in U.S. Pat. No.
5,024,946, by Hagiwara, et al. in U.S. Pat. No. 5,093,261, and by
Anderson, et al. in U.S. Pat. No. 6,753,420 and U.S. Pat. No.
6,417,337; all of which are incorporated herein by reference;
antibodies recognizing tumor associated antigens. Non limiting
examples includes antibodies targeting tumor vasculature
(Duijvestijn et al., J. Immunol., 138(3):713-719, 1987; Hagemeier
et al., Int. J. Cancer, 38:481-488, 1986; Bruland et al., Int. J.
Cancer, 38:27-31, 1986; Murray et al., Radiotherapy and Oncology,
16:221-234, 1989; and Schlingemann et al., Laboratory
Investigation, 52(1):71-76, 1985), and antibodies targeting
tenascin, a large molecular weight extracellular glycoprotein
expressed in the stroma of various benign and malignant tumors
(Shrestha et. al., Eur. J. Cancer B. Oral. Oncol., 30B(6):393-9,
1994; and Tuominen and Kallioinen, J. Cutan. Pathol. 21(5):424-9,
1994.), all of which are incorporated herein by reference;
anti-platelet antibodies described by Gralnick in U.S. Pat. No.
5,366,865, which is incorporated herein by reference; and the use
of streptavidin, avidin, and biotin molecules to conjugate
molecules to one another, to form biotinylated protein molecules,
biotinylated protein-avidin or avidin like complexes, or
multicomponent conjugates, both in vitro and in vivo, is well known
in the Art. See, generally, P. Webber et al., "Science, vol. 243,
pp. 85-88, Jan. 6, 1989", M. Wilchek et al, "Analytical
Biochemistry, vol. 171 pp. 1-32, 1988", Otto C. Boerman et al.,
"Pretargeted Radioimmunotherapy of Cancer: Progress Step by Step",
Journal of Nuclear Medicine Vol. 44 No. 3 400-411, Bayer et al.,
"Trends in Biochemical Science, 3, N257, November 1978", and
Paganelli G, Riva P, Deleide G, et al. "Int J Cancer Suppl. 1988;
2: 121-125", all of which are incorporated herein by reference.
[0009] However, non of these references suggest targeting
anticellular agent-carrying blood platelets to the vasculature of a
solid tumor, and thus inducing the formation of a thrombus within
the tumor vasculature and, at the same time, delivering a high
concentration of an anticellular agent to the tumor cells, and thus
forming a platelet-mediated thrombus within the tumor vasculature
leading to occlusion of the tumor vasculature, with ultimate
destruction of the centrally located tumor cells, followed by
destruction or impairment of the growth or cell division of the
peripherally located tumor cells by the anticellular agent carried
by the blood platelets and concentrated within the tumor.
SUMMARY OF THE INVENTION
[0010] The present invention is directed to and provides, in one
aspect of the invention, a method for treating a vascularized solid
tumor using a plurality of anticellular agent-carrying blood
platelets targeted to the vasculature of the tumor, to induce a
thrombus formation within the tumor vasculature and, at the same
time, to deliver a high concentration of the anticellular agent to
the peripherally located tumor cells, and thus, clearing solid
tumors entirely from a mammalian body.
[0011] The present invention is further directed, in another aspect
of the invention, to means for targeting a plurality of
anticellular agent-carrying blood platelets to a tumor vasculature,
to induce a thrombus formation within the tumor vasculature and, at
the same time, to deliver a high concentration of the anticellular
agent to the peripherally located tumor cells, while avoiding any
deterioration in the functions of the spleen or any other vital
body organ.
[0012] The present invention is further directed, in another aspect
of the invention, to a schedule for treating a mammal affected by
multi-focal secondary metastases deposits of a solid tumor, with at
least one of the secondary metastases deposits being vascularized,
and with at least another one of the secondary metastases deposits
being not-yet vascularized.
[0013] As used herein, the term "parenteral administration" refers
to and includes any route through which a compound is administered
to a mammal other than through the digestive tract, non limiting
examples of such routes include: intravenous injection,
intra-arterial injection, intracavitary injection, intramuscular
injection, and injection through an intravenous line, cannula,
catheter, or the like; the term "parenteral administration of a
sub-therapeutic dose of a small molecular Vascular Disrupting Agent
(VDA)" refers to and includes the administration of any small
molecular Vascular Disrupting Agent (VDA), to a mammal, at any dose
smaller than its minimal therapeutically effective dose, with the
minimal therapeutically effective dose of a VDA being the dose at
or above which the administration of the VDA to the mammal will
result in acute irreversible occlusion of any tumor vasculature;
the term "anti-tumor binding component" refers to and includes any
compound having a binding region specifically binding to an antigen
or a receptor present on the outer surface of a tumor cell, or
present on the outer surface of a component of a tumor associated
vasculature or stroma; the terms "first ligand, second ligand, and
anti-ligand" refers to and includes any complementary set of
molecules that specifically bind to each other; the term
"anti-platelet binding component" refers to and includes any
compound having a binding region specifically binding to an antigen
or a receptor present on the outer surface of a blood platelet; the
term "carrying" refers to and includes either containing the
anticellular agent within the blood platelets, attaching the
anticellular agent to the outer surface of the blood platelet, or
both; the term "anticellular agent" refers to and includes any
agent that destroys, impair the growth or cell division, or
irreversibly alter the metabolism of a cancer cell; the term
"vascularized secondary metastases deposit of a solid tumor" refers
to and includes any secondary metastases deposit of a solid tumor
having its own feeding blood vasculature; and the term "not-yet
vascularized secondary metastases deposit of a solid tumor" refers
to and includes any secondary metastases deposit of a solid tumor
which did not develop its own feeding blood vasculature yet.
[0014] Typical vascularized solid tumors are solid tumors which
require a vascular component for the provision of oxygen and
nutrients. Exemplary solid tumors to which the present invention is
directed, include, but are not limited to, carcinomas of the lung,
breast, ovary, stomach, pancreas, larynx, esophagus, testes, liver,
parotid, biliary tract, colon, rectum, cervix, uterus, endometrium,
kidney, bladder, prostate, thyroid, squamous cell carcinomas,
adenocarcinomas, small cell carcinomas, melanomas, gliomas,
neuroblastomas, different types of sarcomas, and the like.
[0015] Accordingly, the present invention provides method and means
for treating a mammal from a vascularized solid tumor using a
number of, in vitro prepared, anticellular agent-carrying blood
platelets to induce a thrombus formation within the tumor
vasculature, and at the same time to deliver a high concentration
of the anticellular agent to the peripherally located tumor
cells.
[0016] In the method provided in the present invention, a
sub-therapeutic dose of a small molecular Vascular Disrupting Agent
(VDA) is used to selectively disrupt the endothelial lining of the
tumor vasculature, while maintaining adequate rate of blood flow
within the tumor vasculature. Although small molecular VDAs are
tested and developed to provide acute irreversible occlusion of the
tumor vasculature, yet conducted preclinical studies showed that
the administration of a low sub-therapeutic dose of a small
molecular VDA results in disruption of the endothelial lining of
the tumor vasculature, with initial marked reduction in the tumor
blood flow, followed by near complete recovery of the blood flow
within the tumor vasculature after 24 hours. A non limiting example
of these studies is disclosed by Prise V E, Honess D J, Stratford M
R L, Wilson J, Tozer G M., in "The vascular response of tumor and
normal tissues in the rat to the vascular targeting agent,
combretastatin A-4-phosphate, at clinically relevant doses". (Int J
Oncol 2002; 21:717-26.). This study is incorporated herein by
reference.
[0017] In a preferred embodiment of the present invention, the
provided method for treating a vascularized solid tumor includes
the steps of: [0018] a) parenteral administration of a
sub-therapeutic dose of at least one small molecular Vascular
Disrupting Agent (VDA), to disrupt the endothelial lining of the
tumor vasculature, while maintaining adequate rate of blood flow
within the tumor vasculature, and thus, directly exposing the
underlying tumor cells to the circulating blood; [0019] b)
Targeting a number of, in vitro prepared, anticellular agent
carrying blood platelets to the tumor cells exposed in step (a),
leading to immobilization of the said anticellular agent-carrying
blood platelets within the tumor vasculature; [0020] c) allowing
for the induction of a thrombus formation within the tumor
vasculature, leading to occlusion of the tumor vasculature and
destruction of the centrally located tumor cells, followed by
rupture of the said anticellular agent-carrying blood platelets,
with release of their anticellular agent content; and [0021] d)
arranging for the delivery of the anticellular agent released from
the ruptured blood platelets in step (c), to the peripherally
located tumor cells.
[0022] In another preferred embodiment of the present invention,
the provided method for treating a vascularized solid tumor
includes the steps of: [0023] a) parenteral administration of a
sub-therapeutic dose of at least one small molecular Vascular
Disrupting Agent (VDA), to disrupt the endothelial lining of the
tumor vasculature, while maintaining adequate rate of blood flow
within the tumor vasculature, and thus directly exposing the
underlying tumor cells to the circulating blood. In a preferred
embodiment, this step is preceded and/or accompanied by the
administration of at least one anti-coagulant agent, e.g. Heparin,
to prevent the formation of any small thrombi within the tumor
vasculature, which is known to accompany the administration of VDA;
[0024] b) parenteral administration of a number of at least one
type of anti-tumor binding component--first ligand complexes, which
will attach themselves to the exposed tumor cells. In a preferred
embodiment, this step is also preceded and/or accompanied by the
administration of at least one anti-coagulant agent, e.g. Heparin,
to prevent the formation of any small thrombi within the tumor
vasculature; [0025] c) parenteral administration of a number of
anti-ligands, which will attach themselves to the anti-tumor
binding component--first ligand complexes, already attached to the
tumor cells. In a preferred embodiment, this step is also preceded
and/or accompanied by the administration of at least one
anti-coagulant agent, e.g. Heparin, to prevent the formation of any
small thrombi within the tumor vasculature; [0026] d) parenteral
administration of a number of, in vitro prepared, blood platelets,
each blood platelet carrying at least one anticellular agent and
having at least one anti-platelet binding component--second ligand
complex attached to its outer surface. In a preferred embodiment,
this step is also preceded and/or accompanied by the administration
of at least one anti-coagulant agent, e.g. Heparin, to delay the
onset of the self-induced thrombus formation within the tumor
vasculature, as will be described herein after; [0027] e) allowing
the blood platelets to link to the tumor cells through in vivo
formation of anti-tumor binding component--first
ligand--anti-ligand--second ligand--anti-platelet binding component
complexes; [0028] f) parenteral administration of a number of
anti-ligands to allow more anticellular agent-carrying blood
platelets to link to the blood platelets already linked to the
tumor cells, through in vivo formation of anti-platelet binding
component--second ligand--anti-ligand--second ligand--anti-platelet
binding component complexes. In a preferred embodiment, this step
is also preceded and/or accompanied by the administration of at
least one anti-coagulant agent, e.g. Heparin, to delay the onset of
the self-induced thrombus formation within the tumor vasculature,
as will be described herein after, until most of the administered
anticellular agent-carrying blood platelets are immobilized within
the tumor vasculature; [0029] g) allowing for the initiation of the
self-induced thrombus formation within the tumor vasculature, as
will be described herein after, leading to occlusion of the tumor
vasculature and destruction of the centrally located tumor cells,
followed by rupture of the anticellular agent-carrying blood
platelets included within the formed blood thrombus, with release
of their anticellular agent content; and [0030] h) encouraging the
mammal to exercise few times a day, for several days, to evenly
disperse the anticellular agent released from the ruptured blood
platelets within the debris of the centrally located tumor cells,
which enables delivering the anticellular agent to the peripherally
located tumor cells, as will be described herein after.
[0031] In a preferred embodiment, the formation of a blood thrombus
within the tumor vasculature in step (g) is followed by removal of
the freely circulating residual portion of the administered
anticellular agent-carrying blood platelets, which were not
included within the thrombus formed within the tumor vasculature,
from the mammal's blood stream.
[0032] Non limiting examples of small molecular Vascular Disrupting
Agent (VDA) for use with the present invention include the
microtubulin destabilizing drugs, combretastatin A-4 disodium
phosphate, ZD6126, AVE8062, and Oxi 4503; and the flavonoid,
DMXAA.
[0033] Any compound having a binding region specifically binding to
an antigen or a receptor present on the outer surface of a tumor
cell, or present on the outer surface of a component of a tumor
associated vasculature or stroma can be used as the anti-tumor
binding component, according to the present invention. In a
preferred embodiment of the present invention, at least two types
of anti-tumor binding components are used, with one of them
specifically binding to tumor cells, and the other one specifically
binding to a tumor associated vasculature or stroma. Any
complementary set of molecules that specifically bind to each other
can be used as the first ligand, second ligand, and anti-ligand
according to the present invention. Also, any compound having a
binding region specifically binding to an antigen or a receptor
present on the outer surface of a blood platelet can be used as the
anti-platelet binding component according to the present
invention.
[0034] "Platelets" utilized in carrying out the present invention
are, in general, of animal, and preferably mammalian, origin (e.g.,
pig, sheep, cow, horse, goat, cat, dog, mouse, rat, human, etc.).
Platelets may be derived from the same species into which the
platelets are introduced, or from a species different from the
species into which the platelets are introduced. Either freshly
isolated platelets or rehydrated fixed-dried platelets can be used
with the present invention.
[0035] Any agent that destroys, impairs the growth or cell
division, or irreversibly alters the metabolism of cancer cells can
be used as the anticellular agent, according to the present
invention. In a preferred embodiment of the present invention, the
anticellular agent is contained within the blood platelets. In
another preferred embodiment, the anticellular agent is attached to
the outer surface of the blood platelet. In yet another preferred
embodiment, more than one anticellular agent are used, with at
least one anticellular agent being contained within the blood
platelet, and at least one anticellular agent being attached to the
outer surface of the blood platelet.
[0036] In general, the anticellular agent(s) to be delivered is
coupled to or associated with the platelets so that each platelet
carries, or has associated therewith, at least 1,000, and more
preferably at least 10,000, individual molecules of the agent to be
delivered.
[0037] As the life span of the platelets within the formed thrombus
is approximately 10 days, so, everyday about 10% of the platelets
attached to the cancer cells, or to the tumor associated
vasculature or stroma, will rupture spontaneously. The ruptured
platelets will release ADP (Adenosine diphosphate), thromboxane A2,
serotonin, phospholipids, lipoproteins, and other proteins, leading
to the activation of the nearby blood platelets and the initiation
of a blood coagulation cascade. See, for example: Hechler, B.,
Leon, C., Vial, C., Vigne, P., Frelin, C., Cazenave, J. P., and
Gachet, C. (1998) Blood 92, 152-159. The activation of the blood
platelets modifies their membranes in such a way to allow
fibrinogen to adhere to them, which results in attaching the
fibrinogen net of the formed blood thrombus to the outer surface of
the activated blood platelets. And thus, the formed blood thrombus
will be indirectly attached to the tumor cells.
[0038] The formed blood thrombus occludes the blood vessels
in-between the cancer cells, and thus, cutting off the blood supply
to the centrally located cancer cells, leading to their
destruction. This is followed by rupture of the platelets
immobilized at the periphery of the formed blood thrombus, with
release of their anticellular agent content. Then, the patient is
encouraged to exercise few times a day, for several days, to
mechanically agitate the cellular debris within the central part of
the tumor, leading to even dispersion of the anticellular agent,
released from the ruptured blood platelets, within the debris of
the centrally located tumor cells, and thus enabling the
anticellular agent to reach to the intact peripherally located
tumor cells at high concentration, leading to their subsequent
destruction; or impairment of their growth or cellular division; or
irreversibly altering their metabolism, according to the type of
the anticellular agent(s) used, and thus, all the cells of the
solid tumor are destroyed.
[0039] The freely circulating residual portion of the administered
anticellular agent-carrying blood platelets, which were not
included within the thrombus formed within the tumor vasculature,
is either selectively, or non-selectively removed from the mammal's
blood stream, to avoid delivering a high concentration of the used
anticellular agent(s) to the spleen, which will markedly
deteriorate the splenic ability to function properly
afterwards.
[0040] In a preferred embodiment of the present invention, the
provided method is preceded and/or accompanied and/or followed by
conventional enteral or parenteral administration of a therapeutic
dose of at least one anticellular agent, to destroy any small sized
non vascularized solid tumors present within the mammal, as well as
early implanted and not-yet implanted tumor metastases.
[0041] Also, in a preferred embodiment of the present invention,
the provided method is preceded and/or accompanied by the
administration of at least one immuno-suppressive agent to the
mammal, to safe guard against the development of an immune response
against the administered components which will hinder their
re-administration in a following setting, if needed.
[0042] The present invention is further directed, in another aspect
of the invention, to a schedule for treating a mammal affected by
multi-focal, variable-sized, secondary metastases deposits of a
solid tumor, with at least one of the secondary metastases deposits
being vascularized, and with at least another one of the secondary
metastases deposits being not-yet vascularized.
[0043] In a preferred embodiment, the provided schedule for
treating the mammal affected by said multi-focal, variable-sized,
secondary metastases deposits of the solid tumor includes the steps
of: [0044] a) treating the mammal using the method provided in the
present invention for treating a mammal from a vascularized solid
tumor, and described in full details herein before, to destroy the
vascularized secondary metastases deposits of the tumor; [0045] b)
administration of at least one agent to prevent/minimize the
implantation of new tumor secondary metastases deposits within the
mammal's body, for a period of time sufficient for the not-yet
vascularized secondary metastases deposits of the tumor to grow and
develop its own vasculature; and [0046] c) re-treating the mammal
using the method provided in the present invention for treating a
mammal from a vascularized solid tumor, to destroy the now
vascularized secondary metastases deposit(s) of the tumor, and
thus, completely clearing the secondary metastases deposits of the
tumor from the mammal's body.
[0047] In a preferred embodiment, the agent(s) administered to
prevent/minimize the implantation of new tumor secondary metastases
deposits within the mammal's body is selected from the group
consisting of anti-coagulants; platelet inhibitors;
thrombocytopenic agents; and vascular repairing agents.
[0048] These and other aspects of the present invention will become
apparent to those skilled in the art after a reading of the
following description of the preferred embodiment when considered
with the drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0049] The description of the features of the present invention
will be more fully appreciated by reference to the following
detailed description of the exemplary embodiments in accordance
with the accompanying drawings, wherein:
[0050] FIG. 1 is a schematic representation of a multi-step method,
showing the use of anticellular agent-carrying blood platelets,
which are targeted and attached to a tumor vasculature, to treat a
mammal suffering from a solid tumor, according to the present
invention.
[0051] FIG. 2 is a schematic representation of another multi-step
method, showing the use of anticellular agent-carrying blood
platelets, which are targeted and attached to a tumor vasculature,
to treat a mammal suffering from a solid tumor, according to the
present invention.
[0052] FIG. 3 is a schematic representation of another multi-step
method, showing the use of anticellular agent-carrying blood
platelets, which are targeted and attached to a tumor vasculature,
to treat a mammal suffering from a solid tumor, according to the
present invention.
[0053] FIG. 4 is a schematic representation of the sequence with
which the cells of a solid tumor are destroyed, on targeting
anticellular agent-carrying platelets to the tumor vasculature,
according to the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0054] Prior art made of record includes the inventor's earlier
U.S. patent application Ser. Nos. 11/343,694 and 11/399,281, which
provides a method for treating a vascularized solid tumor by
targeting a plurality of, in vitro prepared, anticellular
agent-carrying blood platelets to the vasculature of the tumor, to
induce a thrombus formation within the tumor vasculature, and at
the same time to deliver a high concentration of the anticellular
agent to the tumor cells.
[0055] However, as these before mentioned patent applications don't
provide a definite mechanism with which anti-tumor antibodies may
access the tumor cells, and also don't provide a definite mechanism
with which the anticellular agent carried by the blood platelets
may be provided to the peripherally located tumor cells, so, there
exists a need for a method of treating solid tumors having a
definite mechanism with which anti-tumor antibodies may access the
tumor cells, and having a definite mechanism with which the
anticellular agent carried by the blood platelets may be provided
to the peripherally located tumor cells.
[0056] Also, as these before mentioned patent applications don't
provide a definite mechanism with which relatively small sized, not
yet vascularized, solid tumor metastases deposits may be targeted,
so, there exists a need for a method of treating solid tumors
having a definite mechanism with which small sized, not yet
vascularized, solid tumor metastases deposits may be targeted.
[0057] Accordingly, the present invention is directed to and
provides, in one aspect of the invention, a method for treating a
vascularized solid tumor using a plurality of anticellular
agent-carrying blood platelets targeted to the vasculature of the
tumor, to induce a thrombus formation within the tumor vasculature
and, at the same time, to deliver a high concentration of the
anticellular agent to the peripherally located tumor cells, and
thus, clearing solid tumors entirely from a mammalian body.
[0058] The present invention is further directed, in another aspect
of the invention, to means for targeting a plurality of
anticellular agent-carrying blood platelets to a tumor vasculature,
to induce a thrombus formation within the tumor vasculature and, at
the same time, to deliver a high concentration of the anticellular
agent to the peripherally located tumor cells, while avoiding any
deterioration in the functions of the spleen or any other vital
body organ.
[0059] The present invention is further directed, in another aspect
of the invention, to a schedule for treating a mammal affected by
multi-focal secondary metastases deposits of a solid tumor, with at
least one of the secondary metastases deposits being vascularized,
and with at least another one of the secondary metastases deposits
being not-yet vascularized.
[0060] As used herein, the term "parenteral administration" refers
to and includes any route through which a compound is administered
to a mammal other than through the digestive tract, non limiting
examples of such routes include: intravenous injection,
intra-arterial injection, intracavitary injection, intramuscular
injection, and injection through an intravenous line, cannula,
catheter, or the like; the term "parenteral administration of a
sub-therapeutic dose of a small molecular Vascular Disrupting Agent
(VDA)" refers to and includes the administration of any small
molecular Vascular Disrupting Agent (VDA), to a mammal, at any dose
smaller than its minimal therapeutically effective dose, with the
minimal therapeutically effective dose of a VDA being the dose at
or above which the administration of the VDA to the mammal will
result in acute irreversible occlusion of any tumor vasculature;
the term "anti-tumor binding component" refers to and includes any
compound having a binding region specifically binding to an antigen
or a receptor present on the outer surface of a tumor cell, or
present on the outer surface of a component of a tumor associated
vasculature or stroma; the terms "first ligand, second ligand, and
anti-ligand" refers to and includes any complementary set of
molecules that specifically bind to each other; the term
"anti-platelet binding component" refers to and includes any
compound having a binding region specifically binding to an antigen
or a receptor present on the outer surface of a blood platelet; the
term "carrying" refers to and includes either containing the
anticellular agent within the blood platelets, attaching the
anticellular agent to the outer surface of the blood platelet, or
both; and the term "anticellular agent" refers to and includes any
agent that destroys, impair the growth or cell division, or
irreversibly alter the metabolism of a cancer cell.
[0061] Typical vascularized solid tumors are solid tumors which
require a vascular component for the provision of oxygen and
nutrients. Exemplary solid tumors to which the present invention is
directed, include, but are not limited to, carcinomas of the lung,
breast, ovary, stomach, pancreas, larynx, esophagus, testes, liver,
parotid, biliary tract, colon, rectum, cervix, uterus, endometrium,
kidney, bladder, prostate, thyroid, squamous cell carcinomas,
adenocarcinomas, small cell carcinomas, melanomas, gliomas,
neuroblastomas, different types of sarcomas, and the like.
[0062] Accordingly, the present invention provides method and means
for treating a mammal from a vascularized solid tumor using a
number of, in vitro prepared, anticellular agent-carrying blood
platelets to induce a thrombus formation within the tumor
vasculature, and at the same time to deliver a high concentration
of the anticellular agent to the peripherally located tumor
cells.
[0063] In the method provided in the present invention, a
sub-therapeutic dose of a small molecular Vascular Disrupting Agent
(VDA) is used to selectively disrupt the endothelial lining of the
tumor vasculature, while maintaining adequate rate of blood flow
within the tumor vasculature. Although small molecular VDAs are
tested and developed to provide acute irreversible occlusion of the
tumor vasculature, yet conducted preclinical studies showed that
the administration of a low sub-therapeutic dose of a small
molecular VDA results in disruption of the endothelial lining of
the tumor vasculature, with initial marked reduction in the tumor
blood flow, followed by near complete recovery of the blood flow
within the tumor vasculature after 24 hours. A non limiting example
of these studies is disclosed by Prise V E, Honess D J, Stratford M
R L, Wilson J, Tozer G M., in "The vascular response of tumor and
normal tissues in the rat to the vascular targeting agent,
combretastatin A4-phosphate, at clinically relevant doses". (Int J
Oncol 2002; 21:717-26.). This study is incorporated herein by
reference.
[0064] In a preferred embodiment of the present invention, the
provided method for treating a vascularized solid tumor includes
the steps of: [0065] a) parenteral administration of a
sub-therapeutic dose of at least one small molecular Vascular
Disrupting Agent (VDA), to disrupt the endothelial lining of the
tumor vasculature, while maintaining adequate rate of blood flow
within the tumor vasculature, and thus directly exposing the
underlying tumor cells to the circulating blood; [0066] b)
Targeting a number of in vitro prepared, anticellular
agent-carrying, blood platelets to the tumor cells exposed in step
(a), leading to immobilization of the said anticellular
agent-carrying blood platelets within the tumor vasculature; [0067]
c) allowing for the induction of a thrombus formation within the
tumor vasculature, leading to occlusion of the tumor vasculature
and destruction of the centrally located tumor cells, followed by
rupture of the said anticellular agent-carrying blood platelets,
with release of their anticellular agent content; and [0068] d)
arranging for the delivery of the anticellular agent released from
the ruptured blood platelets in step (c), to the peripherally
located tumor cells.
[0069] In another preferred embodiment of the present invention,
the provided method for treating a vascularized solid tumor
includes the steps of: [0070] a) parenteral administration of a
sub-therapeutic dose of at least one small molecular Vascular
Disrupting Agent (VDA), to disrupt the endothelial lining of the
tumor vasculature, while maintaining adequate rate of blood flow
within the tumor vasculature, and thus directly exposing the
underlying tumor cells to the circulating blood. In a preferred
embodiment, this step is preceded and/or accompanied by the
administration of at least one anti-coagulant agent, e.g. Heparin,
to prevent the formation of any small thrombi within the tumor
vasculature, which is known to accompany the administration of VDA;
[0071] b) parenteral administration of a number of at least one
type of anti-tumor binding component--first ligand complexes, which
will attach themselves to the exposed tumor cells. In a preferred
embodiment, this step is also preceded and/or accompanied by the
administration of at least one anti-coagulant agent, e.g. Heparin,
to prevent the formation of any small thrombi within the tumor
vasculature; [0072] c) parenteral administration of a number of
anti-ligands, which will attach themselves to the anti-tumor
binding component--first ligand complexes, already attached to the
tumor cells. In a preferred embodiment, this step is also preceded
and/or accompanied by the administration of at least one
anti-coagulant agent, e.g. Heparin, to prevent the formation of any
small thrombi within the tumor vasculature; [0073] d) parenteral
administration of a number of, in vitro prepared, blood platelets,
each blood platelet carrying at least one anticellular agent and
having at least one anti-platelet binding component--second ligand
complex attached to its outer surface. In a preferred embodiment,
this step is also preceded and/or accompanied by the administration
of at least one anti-coagulant agent, e.g. Heparin, to delay the
onset of the self-induced thrombus formation within the tumor
vasculature, as will be described herein after; [0074] e) allowing
the blood platelets to link to the tumor cells through in vivo
formation of anti-tumor binding component--first
ligand--anti-ligand--second ligand--anti-platelet binding component
complexes; [0075] f) parenteral administration of a number of
anti-ligands to allow more anticellular agent-carrying blood
platelets to link to the blood platelets already linked to the
tumor cells, through in vivo formation of anti-platelet binding
component--second ligand--anti-ligand--second ligand--anti-platelet
binding component complexes. In a preferred embodiment, this step
is also preceded and/or accompanied by the administration of at
least one anti-coagulant agent, e.g. Heparin, to delay the onset of
the self-induced thrombus formation within the tumor vasculature,
as will be described herein after, until most of the administered
anticellular agent-carrying blood platelets are immobilized within
the tumor vasculature; [0076] g) allowing for the initiation of the
self-induced thrombus formation within the tumor vasculature, as
will be described herein after, leading to occlusion of the tumor
vasculature and destruction of the centrally located tumor cells,
followed by rupture of the anticellular agent-carrying blood
platelets included within the formed blood thrombus, with release
of their anticellular agent content; and [0077] h) encouraging the
mammal to exercise few times a day, for several days, to evenly
disperse the anticellular agent released from the ruptured blood
platelets within the debris of the centrally located tumor cells,
which enables delivering the anticellular agent to the peripherally
located tumor cells, as will be described herein after.
[0078] In a preferred embodiment, the formation of a blood thrombus
within the tumor vasculature in step (g) is followed by removal of
the freely circulating residual portion of the administered
anticellular agent-carrying blood platelets, which were not
included within the thrombus formed within the tumor vasculature,
from the mammal's blood stream.
[0079] Non limiting examples of small molecular Vascular Disrupting
Agent (VDA) for use with the present invention include the
microtubulin destabilizing drugs, combretastatin A4 disodium
phosphate, ZD6126, AVE8062, and Oxi 4503; and the flavonoid, DMXAA.
Such small molecular VDAs and methods for their preparation are
well known to people experienced in the Art.
[0080] Any compound having a binding region specifically binding to
an antigen or a receptor present on the outer surface of a tumor
cell, or present on the outer surface of a component of a tumor
associated vasculature or stroma can be used as the anti-tumor
binding component, according to the present invention. In a
preferred embodiment of the present invention, at least two types
of anti-tumor binding components are used, with one of them
specifically binding to tumor cells, and the other one specifically
binding to a tumor associated vasculature or stroma. Non limiting
examples of anti-tumor binding components for use with the present
invention include: an antibody, a monoclonal antibody, a polyclonal
antibody, a humanized monoclonal antibody, a chimeric antibody, a
single chain antibody, a dimeric single chain antibody construct, a
multimeric single chain antibody construct, a peptide, a nucleic
acid sequence, a protein, a ligand or anti-ligand, an
oligonucleotide, native or naked antibodies; chimeric monoclonal
antibodies; genetically engineered monoclonal antibodies; fragments
of antibodies, tumor-binding peptides; polypeptide; glycoprotein;
lipoprotein, growth factors; lymphokines and cytokines; enzymes,
immune modulators; fusion protein, enzymatic substrate, receptor,
hormone, lectin, cadherin, immunological conjugates, chemical
conjugates, any of the above joined to a molecule that mediates an
effector function; conjugates that include any one of the above,
and fragments or parts of any of the above. Such anti-tumor binding
components and methods for their preparation are well known to
people experienced in the Art.
[0081] Any complementary set of molecules that specifically bind to
each other can be used as the first ligand, second ligand, and
anti-ligand according to the present invention. Non limiting
examples of such complementary sets of molecules for use with the
present invention include: biotin/avidin or streptavidin or a
chemically modified form of streptavidin or avidin, zinc finger
protein/dsDNA fragment, enzyme/inhibitor, hapten/antibody,
ligand/receptor, homophylic peptides and leucine zipper sets. Such
complementary sets of molecules and methods for their preparation
are well known to people experienced in the Art. See, generally, P.
Webber et al., "Science, vol. 243, pp. 85-88, Jan. 6, 1989", M.
Wilchek et al, "Analytical Biochemistry, vol. 171 pp. 1-32, 1988",
Bayer et al., "Trends in Biochemical Science, 3, N257, November
1978", and Paganelli G, Riva P, Deleide G, et al. "Int J Cancer
Suppl. 1988; 2: 121-125"; all of which are incorporated herein by
reference.
[0082] Also, any compound having a binding region specifically
binding to an antigen or a receptor present on the outer surface of
a blood platelet can be used as the anti-platelet binding component
according to the present invention. Any compound having a binding
region specifically binding to an antigen or a receptor present on
the outer surface of a blood platelet can be used as the
anti-platelet binding component according to the present invention.
Non limiting examples of anti-platelet binding components for use
with the present invention include: anti-platelet monoclonal
antibodies described by Gralnick in U.S. Pat. No. 5,366,865, von
Willebrand factor, osteopontin, fibrinogen, fibrin, fibronectin,
vitronectin, collagen, thrombospondin, laminin, heparin, heparan
sulfate, chondroitin sulfate, phospholipase A2, matrix
metalloproteinases, thrombin, glass, sialyl-lewis X, fibulin-1,
PECAM, ICAM-1, ICAM-2, p-selectin ligand, MAC-1, LFA-1, portions of
any of the above, and functional equivalents of any of the above.
Such anti-platelet binding components and methods for their
preparation are well known to people experienced in the Art.
[0083] "Platelets" utilized in carrying out the present invention
are, in general, of animal, and preferably mammalian, origin (e.g.,
pig, sheep, cow, horse, goat, cat, dog, mouse, rat, human, etc.).
Platelets may be derived from the same species into which the
platelets are introduced, or from a species different from the
species into which the platelets are introduced. In a preferred
embodiment, platelets are harvested from a subject, prepared
according to the method provided in the present invention, and
after being so prepared are administered at a later time back to
the same subject from which the platelets were harvested.
[0084] Either freshly isolated platelets or rehydrated fixed-dried
platelets can be used with the present invention. In a preferred
embodiment, platelets are freshly isolated, prepared according to
the method provided in the present invention, and then
administered, either to the same subject from whom it was
harvested, or to another subject.
[0085] In another preferred embodiment, fixed-dried platelets are
used, with the anticellular agent(s) and the anti-platelet binding
component--second ligand complexes being attached and/or
internalized into the platelets either before or after fixing
and/or drying the platelets, and on a later time the platelets are
rehydrated and administered either to the same subject from whom it
was harvested, or to another subject. The use of fixed-dried
platelets enables safe extension of the time period between the
harvesting of the platelets and their administering. Platelets may
be fixed in accordance with known techniques, such as described in
U.S. Pat. Nos. 4,287,087; 5,651,966; 5,902,608; 5,891,393; and
5,993,084. Drying of platelets after fixation may be carried out by
any suitable means, such as lyophilization.
[0086] Any agent that destroys, impairs the growth or cell
division, or irreversibly alters the metabolism of cancer cells can
be used as the anticellular agent, according to the present
invention. Non limiting examples of anti-cellular agents for use
with the present invention include: cytotoxins, chemotherapeutic
agents; steroids, antimetabolites, anthracyclines, vinca alkaloids,
antibiotics, alkylating agents, epipodophyllotoxins; any plant-,
fungus- or bacteria-derived agent; and radioactive isotopes such as
.sup.125I, .sup.131I, and .sup.86Rb,
[0087] In a preferred embodiment of the present invention, the
anticellular agent(s) is internalized into the blood platelets,
using any suitable technique. Non limiting examples of the
techniques with which an anticellular agent may be internalized
into the platelets are: (1) conjugating the compound to be
delivered to a polymer that is in turn coupled to the platelet's
internal membrane; (2) incorporating the compound to be delivered
into unilamellar or multilamellar phospholipid vesicles that are in
turn internalized into the platelets; (3) absorbing or
internalizing the compound to be delivered into nanoparticles,
e.g., buckminsterfullerene, that are in turn internalized into the
platelets; (4) coupling the compound to be delivered to proteins
that are internalized for trafficking to alpha granules in the
platelets; (5) coupling the compound to be delivered to proteins
(or other macromolecules) or particles that are phagocitized by the
platelets; (6) adsorbing the compound to the exterior surface of
the cell by non-covalent physical or chemical adsorption, that are
in turn internalized into the platelets; and (7) physically
entrapping the compound to be delivered in the platelet
intracellular space through pores that are formed with
electroporation, complement treatment, lytic protein exposure, and
the like. These and other techniques used for intraluminal delivery
of drugs are well known to people experienced in the Art.
[0088] In another preferred embodiment, the anticellular agent(s)
is attached to the outer surface of the blood platelet, using any
suitable technique. Non limiting examples of the techniques with
which an anticellular agent may be attached to the outer surface of
the platelets are: (1) directly chemically coupling the compound to
be delivered to the platelet surface membrane; (2) attaching the
anticellular agent to an anti-platelet binding component, e.g. an
antibody or a ligand, which attaches to an antigen or a receptor on
the outer surface of the blood platelets; and (3) adsorbing the
compound to the exterior surface of the cell by non-covalent
physical or chemical adsorption. These and other techniques used
for attaching a compound to the outer surface of platelets or cells
are well known to people experienced in the Art.
[0089] In yet another preferred embodiment, more than one
anticellular agent are used, with at least one anticellular agent
being internalized into the blood platelet, and at least one
anticellular agent being attached to the outer surface of the blood
platelet, using any combination of the techniques described herein
above.
[0090] In general, the anticellular agent(s) to be delivered is
coupled to or associated with the platelets so that each platelet
carries, or has associated therewith, at least 1,000, and more
preferably at least 10,000, individual molecules of the agent to be
delivered.
[0091] As the life span of the platelets within the formed thrombus
is approximately 10 days, so, everyday about 10% of the platelets
attached to the cancer cells, or to the tumor associated
vasculature or stroma, will rupture spontaneously. The ruptured
platelets will release ADP (Adenosine diphosphate), thromboxane A2,
serotonin, phospholipids, lipoproteins, and other proteins, leading
to the activation of the nearby blood platelets and the initiation
of a blood coagulation cascade. See, for example: Hechler, B.,
Leon, C., Vial, C., Vigne, P., Frelin, C., Cazenave, J. P., and
Gachet, C. (1998) Blood 92, 152-159. The activation of the blood
platelets modifies their membranes in such a way to allow
fibrinogen to adhere to them, which results in attaching the
fibrinogen net of the formed blood thrombus to the outer surface of
the activated blood platelets. And thus, the formed blood thrombus
will be indirectly attached to the tumor cells.
[0092] The formed blood thrombus occludes the blood vessels
in-between the cancer cells, and thus, cutting off the blood supply
to the centrally located cancer cells, leading to their
destruction. This is followed by rupture of the platelets
immobilized at the periphery of the formed blood thrombus, with
release of their anticellular agent content. Then, the patient is
encouraged to exercise few times a day, for several days, to
mechanically agitate the cellular debris within the central part of
the tumor, leading to even dispersion of the anticellular agent,
released from the ruptured blood platelets, within the debris of
the centrally located tumor cells, and thus enabling the
anticellular agent to reach to the intact peripherally located
tumor cells at high concentration, leading to their subsequent
destruction; or impairment of their growth or cellular division; or
irreversibly altering their metabolism, according to the type of
the anticellular agent(s) used, and thus, all the cells of the
solid tumor are destroyed.
[0093] The freely circulating residual portion of the administered
anticellular agent-carrying blood platelets, which were not
included within the thrombus formed within the tumor vasculature,
is non-selectively removed from the mammal's blood stream using the
well known apheresis procedure. In another preferred embodiment,
the freely circulating residual portion of the administered
anticellular agent-carrying blood platelets, which were not
included within the thrombus formed within the tumor vasculature,
is selectively removed from the mammal's blood stream using sheet
membranes, hollow fibers, or packed beds of either beads or
particles having physically adsorbed or covalently attached
anti-ligands. The anti-ligands will selectively bind the
anticellular agent-carrying blood platelets through the formation
anti-ligand--second ligand--anti-platelet binding component
complexes, and thus, selectively removing the freely circulating
residual portion of the administered blood platelets from the
mammal's blood stream. Such techniques and means for their
conduction are well known to people experienced in the Art.
[0094] In a preferred embodiment of the present invention, the
provided method is preceded and/or accompanied and/or followed by
conventional enteral or parenteral administration of a therapeutic
dose of at least one anticellular agent, to destroy any small sized
non vascularized tumors present within the mammal, as well as early
implanted and not-yet implanted tumor metastases.
[0095] Also, in a preferred embodiment of the present invention,
the provided method is preceded and/or accompanied by the
administration of at least one immuno-suppressive agent to the
mammal, to safe guard against the development of an immune response
against the administered components which will hinder their
re-administration in a following setting, if needed. Non-limiting
examples of immunosuppressive agents for use with the present
invention includes: alkylating agents such as cyclophosamide;
nucleic acid antimetabolites such as 6-mercaptopurine and
azathiopurine; antibiotics such as mitomycin C; steroids; folic
acid antagonists such as methotrexate; and plant alkaloids such as
colchicine and vinblastine; and cyclic polypeptides such as
cyclosporine. Such immunosuppressive agents are well known to
people experienced in the Art.
[0096] The present invention is further directed, in another aspect
of the invention, to a schedule for treating a mammal affected by
multi-focal, variable-sized, secondary metastases deposits of a
solid tumor, with at least one of the secondary metastases deposits
being vascularized, and with at least another one of the secondary
metastases deposits being not-yet vascularized.
[0097] In a preferred embodiment, the provided schedule for
treating the mammal affected by said multi-focal, variable-sized,
secondary metastases deposits of the solid tumor includes the steps
of: [0098] a) treating the mammal using the method provided in the
present invention for treating a mammal from a vascularized solid
tumor, and described in full details herein before, to destroy the
vascularized secondary metastases deposits of the tumor; [0099] b)
administration of at least one agent to prevent/minimize the
implantation of new tumor secondary metastases deposits within the
mammal's body, for a period of time sufficient for the not-yet
vascularized secondary metastases deposits of the tumor to grow and
develop its own vasculature; and [0100] c) re-treating the mammal
using the method provided in the present invention for treating a
mammal from a vascularized solid tumor, to destroy the now
vascularized secondary metastases deposit(s) of the tumor, and
thus, completely clearing the secondary metastases deposits of the
tumor from the mammal's body.
[0101] In a preferred embodiment, the agent(s) administered to
prevent/minimize the implantation of new tumor secondary metastases
deposits within the mammal's body is selected from the group
consisting of anti-coagulants, e.g. Vitamin K antagonists such as
Dicumarol, Warfarin, or others; platelet inhibitors;
thrombocytopenic agents; and vascular repairing agents. The role of
such agents in minimizing/preventing the implantation of new tumor
secondary metastases deposits within a mammal's body has been
confirmed in different studies conducted to explore the role of
blood platelets in tumor metastases. A non limiting example of
these studies is disclosed by Gabriel J. Gasic in "Role of plasma,
platelets, and endothelial cells in tumor metastases" (Cancer and
Metastases Reviews, volume 3, Number 2/June 1984: 99-114). This
study is incorporated herein by reference.
[0102] These and other aspects of the present invention will become
apparent to those skilled in the art after a reading of the
following description of the preferred embodiment when considered
with the drawings.
[0103] In the following preferred embodiments the "anti-tumor
binding component--first ligand complex" is exemplified by a
"biotinylated anti-tumor antibody"; the "anti-ligand" is
exemplified by "avidin, or avidin like molecules"; and the
"anti-platelet binding component--second ligand complex" is
exemplified by a "biotinylated anti-platelet antibody", with
freshly isolated blood platelets being used as the vehicle through
which the anticellular agents are being delivered. These components
and techniques are used as illustrative examples, and are not
intended to limit the scope of the compounds and techniques that
may be used according to the present invention.
FIG. 1 is a schematic representation of a multi-step method,
showing the use of anticellular agent-carrying blood platelets,
which are targeted and attached to a tumor vasculature, to treat a
mammal suffering from a solid tumor, according to the present
invention. The provided method includes the steps of: [0104] a)
parenteral administration of a sub-therapeutic dose of a small
molecular Vascular Disrupting Agent (VDA) (11), to disrupt the
endothelial lining (12) of the tumor vasculature, while maintaining
adequate rate of blood flow within the tumor vasculature, and thus
directly exposing the underlying tumor cells (13) to the
circulating blood; [0105] b) parenteral administration (14) of a
number of biotinylated anti-tumor antibodies (15), which will
attach themselves to the tumor (16); [0106] c) parenteral
administration (17) of a number of avidin or avidin like molecules
(18), which will attach themselves (19) to the biotinylated
anti-tumor antibodies already attached to the tumor, along with
attaching any free circulating non implanted tumor metastases to
the main tumor bulk (not shown in the drawing for simplicity);
[0107] d) parenteral administration (20) of a number of, in vitro
prepared, blood platelets (21), each blood platelet has at least
one anticellular agent (22) and at least one biotinylated
anti-platelet antibody (23) attached to its outer surface. The in
vitro preparation of the administered blood platelets includes the
steps of: collecting a number of blood platelets (24), either from
the same mammal or from an immunologically compatible mammal using,
for example, the well known apheresis procedure; attaching the used
anticellular agent (22) to the outer surface of the platelets using
one of the techniques described herein above; and incubating the
collected blood platelets in a solution having biotinylated
anti-platelet antibodies (23) within it for a time sufficient for
the complexes to attach to the outer surface of the platelets (21);
[0108] e) allowing the blood platelets to link to the tumor
vasculature through in vivo formation of biotin-avidin-biotin or
biotin-avidin like-biotin linkages (25) with the biotinylated
anti-tumor antibodies already attached to the tumor; [0109] f)
parenteral administration (26) of a number avidin or avidin like
molecules (27), to allow more blood platelets (28) to link to the
blood platelets (29) already linked to the tumor vasculature
through in vivo formation of biotin-avidin-biotin or biotin-avidin
like-biotin linkages (30) with the biotinylated anti-platelet
antibodies attached to the surfaces of the platelets already
attached to the tumor, with this step being accompanied by The
administration of at least one anti-coagulant agent, e.g. Heparin,
to delay the onset of the self-induced thrombus formation within
the tumor vasculature, as will be described herein before, until
most of the administered anticellular agent-carrying blood
platelets are immobilized within the tumor vasculature; [0110] g)
stopping the anti-coagulant administration to allow for the
initiation of the self-induced thrombus formation within the tumor
vasculature, as will be described herein before; [0111] h) removing
the freely circulating residual portion of the administered
anticellular agent-carrying blood platelets, which were not
included within the thrombus formed within the tumor vasculature,
from the mammal's blood stream. This step is not shown in the
drawing for simplicity. [0112] i) encouraging the mammal to
exercise few times a day, for several days, to evenly disperse the
anticellular agent released from the ruptured blood platelets
within the debris of the centrally located tumor cells, which
enables delivering the anticellular agent to the peripherally
located tumor cells, as will be described herein after. FIG. 2 is a
schematic representation of another multi-step method, showing the
use of anticellular agent-carrying blood platelets, which are
targeted and attached to the tumor vasculature, to treat a mammal
suffering from a solid tumor, according to the present invention.
The provided method includes the steps of: [0113] a) parenteral
administration of a sub-therapeutic dose of a small molecular
Vascular Disrupting Agent (VDA) (31), to disrupt the endothelial
lining (32) of the tumor vasculature, while maintaining adequate
rate of blood flow within the tumor vasculature, and thus directly
exposing the underlying tumor cells (33) to the circulating blood;
[0114] b) parenteral administration (34) of a number of
biotinylated anti-tumor antibodies (35), which will attach
themselves to the tumor (36); [0115] c) parenteral administration
(37) of a number of avidin or avidin like molecules (38), which
will attach themselves (39) to the biotinylated anti-tumor
antibodies already attached to the tumor, along with attaching any
free circulating non implanted tumor metastases to the main tumor
bulk (not shown in the drawing for simplicity); [0116] d)
parenteral administration (40) of a number of, in vitro prepared,
blood platelets (41), each blood platelet has at least one
anticellular agent (42) and at least one biotinylated anti-platelet
antibody (43) attached to its outer surface. The in vitro
preparation of the administered blood platelets includes the steps
of: collecting a number of blood platelets (44), either from the
same mammal or from an immunologically compatible mammal using, for
example, the well known apheresis procedure; internalizing the used
anticellular agent (42) inside the blood platelets using one of the
techniques described herein above; and incubating the collected
blood platelets in a solution having biotinylated anti-platelet
antibodies (43) within it for a time sufficient for the complexes
to attach to the outer surface of the platelets (41); [0117] e)
allowing the blood platelets to link to the tumor vasculature
through in vivo formation of biotin-avidin-biotin or biotin-avidin
like-biotin linkages (45) with the biotinylated anti-tumor
antibodies already attached to the tumor; [0118] f) parenteral
administration (46) of a number avidin or avidin like molecules
(47), to allow more blood platelets (48) to link to the blood
platelets (49) already linked to the tumor vasculature through in
vivo formation of biotin-avidin-biotin or biotin-avidin like-biotin
linkages (50) with the biotinylated anti-platelet antibodies
attached to the surfaces of the platelets already attached to the
tumor, with this step being accompanied by The administration of at
least one anti-coagulant agent, e.g. Heparin, to delay the onset of
the self-induced thrombus formation within the tumor vasculature,
as will be described herein before, until most of the administered
anticellular agent-carrying blood platelets are immobilized within
the tumor vasculature; [0119] g) stopping the anti-coagulant
administration to allow for the initiation of the self-induced
thrombus formation within the tumor vasculature, as will be
described herein before; [0120] h) removing the freely circulating
residual portion of the administered anticellular agent-carrying
blood platelets, which were not included within the thrombus formed
within the tumor vasculature, from the mammal's blood stream. This
step is not shown in the drawing for simplicity. [0121] i)
encouraging the mammal to exercise few times a day, for several
days, to evenly disperse the anticellular agent released from the
ruptured blood platelets within the debris of the centrally located
tumor cells, which enables delivering the anticellular agent to the
peripherally located tumor cells, as will be described herein
after. FIG. 3 is a schematic representation of another multi-step
method, showing the use of anticellular agent-carrying blood
platelets, which are targeted and attached to the tumor
vasculature, to treat a mammal suffering from a solid tumor,
according to the present invention. The provided method includes
the steps of: [0122] a) parenteral administration of a
sub-therapeutic dose of a small molecular Vascular Disrupting Agent
(VDA) (51), to disrupt the endothelial lining (52) of the tumor
vasculature, while maintaining adequate rate of blood flow within
the tumor vasculature, and thus directly exposing the underlying
tumor cells (53) to the circulating blood; [0123] b) parenteral
administration (54) of a number of biotinylated anti-tumor
antibodies (55), which will attach themselves to the tumor (56);
[0124] c) parenteral administration (57) of a number of avidin or
avidin like molecules (58), which will attach themselves (59) to
the biotinylated anti-tumor antibodies already attached to the
tumor, along with attaching any free circulating non implanted
tumor metastases to the main tumor bulk (not shown in the drawing
for simplicity); [0125] d) parenteral administration (60) of a
number of, in vitro prepared, blood platelets (61), each blood
platelet has at least one anticellular agent within its cavity
(62), and at least one anticellular agent (63) and at least one
biotinylated anti-platelet antibody (64) attached to its outer
surface. The in vitro preparation of the administered blood
platelets includes the steps of: collecting a number of blood
platelets (65), either from the same mammal or from an
immunologically compatible mammal using, for example, the well
known apheresis procedure; internalizing one of the used
anticellular agents (62) inside the blood platelets and attaching
the other anticellular agent (63) to the outer surface of the
platelets using any combination of the techniques described herein
above; and incubating the collected blood platelets in a solution
having biotinylated anti-platelet antibodies (64) within it for a
time sufficient for the complexes to attach to the outer surface of
the platelets (61); [0126] e) allowing the blood platelets to link
to the tumor vasculature through in vivo formation of
biotin-avidin-biotin or biotin-avidin like-biotin linkages (66)
with the biotinylated anti-tumor antibodies already attached to the
tumor; [0127] f) parenteral administration (67) of a number avidin
or avidin like molecules (68), to allow more blood platelets (69)
to link to the blood platelets (70) already linked to the tumor
vasculature through in vivo formation of biotin-avidin-biotin or
biotin-avidin like-biotin linkages (71) with the biotinylated
anti-platelet antibodies attached to the surfaces of the platelets
already attached to the tumor, with this step being accompanied by
The administration of at least one anti-coagulant agent, e.g.
Heparin, to delay the onset of the self-induced thrombus formation
within the tumor vasculature, as will be described herein before,
until most of the administered anticellular agent-carrying blood
platelets are immobilized within the tumor vasculature; [0128] g)
stopping the anti-coagulant administration to allow for the
initiation of the self-induced thrombus formation within the tumor
vasculature, as will be described herein before; [0129] h) removing
the freely circulating residual portion of the administered
anticellular agent-carrying blood platelets, which were not
included within the thrombus formed within the tumor vasculature,
from the mammal's blood stream. This step is not shown in the
drawing for simplicity. [0130] i) encouraging the mammal to
exercise few times a day, for several days, to evenly disperse the
anticellular agent released from the ruptured blood platelets
within the debris of the centrally located tumor cells, which
enables delivering the anticellular agent to the peripherally
located tumor cells, as will be described herein after.
FIG. 4 is a schematic representation of the sequence with which the
cells of a solid tumor are destroyed, on targeting anticellular
agent-carrying platelets to the tumor vasculature, according to the
present invention.
[0131] For illustrative purposes, as shown in step (a), the tumor
cells are divided into two portions: a first portion comprising
centrally located tumor cells (81), which depends for their
nutrition on the tumor vasculature (82); and a second portion
comprising peripherally located tumor cells (83), which depend for
their nutrition on the surrounding blood vessels (84) and
surrounding interstitial fluid (85).
[0132] The platelet-mediated thrombus formed within the tumor
vasculature (86) leads to occlusion of the tumor vasculature (82),
with ultimate destruction of the centrally located tumor cells
(87), and as shown in step (b). This is followed by rupture of the
anticellular agent-carrying blood platelets immobilized at the
periphery of the formed blood thrombus (86), with release of their
anticellular agent content. Then, the patient is encouraged to
exercise few times a day, for several days, to mechanically agitate
the cellular debris within the central part of the tumor, leading
to even dispersion of the anticellular agent, released from the
ruptured blood platelets, within the debris of the centrally
located tumor cells (87), and thus enabling the anticellular agent
to reach to the intact peripherally located tumor cells (88) at
high concentration, leading to their subsequent destruction (89);
or impairment of their growth or cellular division; or irreversibly
altering their metabolism, according to the type of the
anticellular agent(s) used, as shown in step (c), and thus, all the
cells of the solid tumor are destroyed.
[0133] Certain modifications and improvements will occur to those
skilled in the art upon a reading of the detailed description. All
modifications and improvements have been deleted herein for the
sake of conciseness and readability but are properly within the
scope of the following claims.
* * * * *